The autoantibody response in systemic autoimmune diseases focuses on a specific set of autoantigens, but it is not clear why certain molecules, such as nuclear antigens and self-IgG are targets in autoimmune diseases. Normally, immune responses require inflammatory and/or costimulatory signals to be initiated. Although autoimmune responses resemble normal immune responses, it is not clear how such signals derive in the case of spontaneous autoimmunity. Recently, it was demonstrated by the Rothstein and Shlomchik labs that activation of rheumatoid factor (RF)-specific B cells by their antigenic immune-complexes was greatly potentiated if chromatin was contained in these complexes, and this was dependent on intact Toll-like receptor (TLR) signaling, most likely via TLR9, a receptor for DNA. These in vitro findings have led to the central hypothesis of this proposal: that certain autoantigens, in particular chromatin, can be ligands for TLRs or other innate immune response receptors, and that the ligation of such receptors by certain autoantigens can play a critical role in autoimmune responses of B cells. We will focus on testing this hypothesis on the role of TLRs in vivo. In the first Aim we will use genetic approaches in vivo to test the role of candidate receptors in influencing autoimmunity. We will then investigate their mechanism of action: In Aim 2 we will use mice transgenic (Tg) for autoreactive BCRs to determine how implicated TLRs act to promote autoimmunity. In particular, we will use the RF Tg mouse system, which has been extensively studied in our lab. There is striking spontaneous activation of RF B cells on the autoimmune-prone MRL/lpr background. We will also study well-characterized Ig-Tg specific for chromatin and Sm. The use of the Ig-Tg systems will allow us to determine the checkpoints that are influenced by TLRs with much greater resolution than possible in polyclonal systems. In Aim 3 we will investigate the role of TLR signaling in the B cell per se. Because TLR9 is already strongly implicated in the activation of RF B cells in vitro and also by emerging data in vivo, we will focus on the role of this receptor on B cells.